Methylated Gene Biomarkers For Detecting Cancer

ABSTRACT

The present invention includes methods diagnosising of cancer by analysis of a patient sample, particularly for the presence of a methylated SPARC nucleic acid molecule, and particularly for the diagnosis of pancreatic cancer. The invention also includes therapeutic methods for treating cancers by administering to cancers patients therapeutically effective amounts of demethylating agents.

The provisional application U.S. Ser. No. 60/482,146 filed Jun. 24, 2003is incorporated herein, by reference, in its entirety.

FIELD OF THE INVENTION

The invention provides for methylated gene biomarkers important in thedetection of cancer. More particularly, the present invention relates toa biomarker which is a methylated gene for SPARC.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Full citations for those references that arenumbered can be found at the end of the specification. Each citation isincorporated herein as though set forth in full.

Pancreatic cancer continues to have one of the highest mortality ratesof any malignancy. Each year, 28,000 patients are diagnosed withpancreatic cancer, and most will die of the disease. The vast majorityof patients are diagnosed at an advanced stage of disease becausecurrently no tumor markers are known that allow reliable screening forpancreas cancer at an earlier, potentially curative stage. This is aparticular problem for those patients with a strong familial history ofpancreatic cancer, who may have up to a 5-7 fold greater risk ofdeveloping pancreatic cancer in their lifetime. Despite several advancesin our basic understanding and clinical management of pancreatic cancer,virtually all patients who will be diagnosed with pancreatic cancer willdie from this disease. The high mortality of pancreatic cancer ispredominantly due to consistent diagnosis at an advanced stage ofdisease, and a lack of effective screening methods.

Infiltrating ductal adenocarcinoma of the pancreas is one of the mostaggressive of all of the solid neoplasms, and invasive pancreatic canceris often associated with a prominent host desmoplastic response. Besidesthe potential aggressiveness of neoplastic cells themselves, this hostresponse at the site of primary invasion has been considered animportant factor in pancreatic cancer progression. Indeed, evidenceexists for interactions between pancreatic cancer cells and stromalfibroblasts that affect the invasive phenotype of pancreatic cancer(Maehara et al., 2001). In contrast to the substantial progress in ourunderstanding of the genetic and epigenetic events that occur withinpancreatic cancer cells, molecular mechanisms associated with thetumor-host interactions have not been well characterized. Ryu andcolleagues used serial analysis of gene expression (SAGE) to comparegene expression profiles of primary carcinomas and passaged cancer celllines, and identified a cluster of invasion-specific genes (Ryu et al.,2001). Many of the genes identified were expressed specifically bystromal cells adjacent to the neoplastic epithelium, thus representingpotential mediators of the tumor-host interactions (Iacobuzio-Donahue etal., 2002b).

SPARC (secreted protein acidic and rich in cysteine)/osteonectin/BM 40is a matricellular glycoprotein involved in diverse biologicalprocesses, including tissue remodeling, wound repair, morphogenesis,cellular differentiation, cell proliferation; cell migration, andangiogenesis (Jendraschak and Sage, 1996; Yan and Sage, 1999; Bradshawand Sage, 2001; Brekken and Sage, 2001). SPARC is highly expressed in awide range of human malignant neoplasms, and the deregulated expressionof SPARC is often correlated with disease progression and/or poorprognosis (Wewer et al., 1988; Bellahcene and Castronovo, 1995; Porte etal., 1995; Porter et al., 1995; Ledda et al., 1997; Porte et al., 1998;Massi et al., 1999; Rempel et al., 1999; Thomas et al., 2000; Yamanakaet al., 2001). Interestingly, in certain tumor types, strong expressionof SPARC has been detected predominantly in the stroma adjacent to theneoplastic cells (Le Bail et al., 1999; Paley et al., 2000;Iacobuzio-Donahue et al., 2002a). These findings have led to thehypothesis that SPARC plays a role in tumor progression at the site ofinterface between neoplastic cells and the surrounding host cells.Recently, Yiu and coworkers have shown that treatment of ovarian cancercells with exogenous SPARC inhibits cell proliferation and inducesapoptosis (Yiu et al., 2001). In addition, forced expression of SPARC inovarian cancer cells resulted in reduced tumorigenicity in nude mice,suggesting that SPARC has a tumor-suppressor function (Mok et al.,1996). In addition to its effects on cellular proliferation, SPARC hasbeen linked with tumor invasion. SPARC has been shown to increase theinvasive capacity of prostate and breast cancer cells in vitro (Jacob etal., 1999; Briggs et al., 2002) and promote invasion of glioma in vivo(Schultz et al., 2002). Thus, the biological functions of SPARC appearto be variable among cancer types, and it is not known whether thisprotein is involved in pancreatic cancer progression.

There is an urgent need, therefore, to determine SPARC's exact role inpancreatic cancer and other types of cancer. Furthermore, there is alsoa great need for the development of new methods for detection anddiagnosis of pancreatic cancers, particularly at a pre-invasive or earlystage of the disease so that early medical intervention can be moreeffective at saving lives. Indeed, new methods of detection forpancreatic cancer may be useful in diagnosing other types of cancer, aswell.

SUMMARY OF THE INVENTION

The invention provides methods for the detection of cancer, inparticular pancreatic cancer, at an early stage of the disease that canallow for early medical treatment and enhanced patient survival rates.

The present invention relates to methods for diagnosing cancer,comprising the detection of a methylated SPARC nucleic acid molecule ora variant thereof in a sample from a subject. The method of theinvention includes modification of SPARC DNA by sodium bisulfite or acomparable agent which converts all unmethylated but not methylatedcytosines to uracil, and subsequent amplification with primers specificfor methylated versus unmethylated DNA. This method of “methylationspecific PCR” or MSP, requires only small amounts of DNA, is sensitiveto 0.1% of methylated alleles of a given CpG island locus, and can bepreformed from a variety of sample types.

The presence of the methylated SPARC nucleic acid molecules iscorrelated to a sample of a normal subject. The sample is preferablyobtained from a mammal suspected of having a proliferative cell growthdisorder, in particular, a pancreatic cancer.

In a preferred embodiment a nucleic acid molecule that is indicative ofa pancreatic cancer comprises a sequence having at least about 80%sequence identity to a molecule identified in SEQ ID NO: 1 (SPARCnucleic acid sequence), more preferably the nucleic acid moleculecomprises a sequence having at least about 90% sequence identity to amolecule identified in SEQ ID NO: 1, most preferably the nucleic acidmolecule comprises a sequence having at least about 95% sequenceidentity to a molecule identified in SEQ ID NO: 1.

In another preferred embodiment, the nucleic acid molecule is expressedat a lower level in a patient with cancer as compared to expressionlevels in a normal individual. Preferably the nucleic acid molecule isexpressed at least about 15 fold lower in a patient with cancer ascompared to expression in a normal individual, more preferably thenucleic acid molecule is expressed at least about 10 fold lower in apatient with cancer as compared to expression in a normal individual,most preferably the nucleic acid molecule is expressed at least about 5fold lower in a patient with cancer as compared to expression in anormal individual.

In another preferred embodiment, the sample used for detection ofpreferred nucleic acid molecules is obtained from a mammalian patient,including a human patient.

The invention also provides methods for treating a mammal suffering fromcancer comprising administering to the mammal a therapeuticallyeffective amount of a demethylating agent. The method can be used totreat a patient is suffering from a pancreatic cancer.

Diagnostic kits are also provided comprising a molecule substantiallycomplementary to a sequence corresponding to a molecule identified inSEQ ID NO: 1. Preferably, the kit comprises a molecule comprising asequence having at least about 80% sequence identity to a moleculeidentified in SEQ ID NO: 1, more preferable at least about 90% sequenceidentity to a molecule identified in SEQ ID NO: 1, most preferable thekit comprises a molecule comprising a sequence having at least about 95%sequence identity to a molecule identified in SEQ ID NO: 1.

Preferably, the kit comprises written instructions for use of the kitfor detection of cancer and the instructions provide for detectingmethylated SPARC nucleic acid molecules from cancer patients.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents (a) Online SAGE Tag to Gene Mapping analysisdemonstrating the frequency of the Hs.111779 tag (ATGTGAAGAG)corresponding to the SPARC gene in 8 pancreatic SAGE libraries derivedfrom short-term cultures of normal pancreatic ductal epithelial cells(H126 and HX), pancreatic cancer cell lines (CAPAN1, CAPAN2, HS766T, andPanc1), and primary pancreatic adenocarcinoma tissue (Panc 91-16113 andPanc 96-6252); (b) Gene expression analysis of SPARC by oligonucleotidemicroarrays in two frozen tissue samples of normal pancreatic ductalepithelial cells selectively microdissected by LCM, a non-neoplasticpancreatic epithelial cell line (HPDE), and 5 pancreatic cancer celllines (AsPC1, CFPAC1, Hs766T, MiaPaCa2, and Panc1); (c) Reversetranscription-PCR analysis of SPARC in a non-neoplastic pancreatic ductepithelial cell line (HPDE), primary fibroblasts derived from pancreaticcancer, and 17 pancreatic cancer cell lines; glyceraldehyde-3-phosphatedehydrogenase (GAPDH) serves as an RNA control.

FIG. 2 represents immunohistochemical staining for SPARC in pancreaticadenocarcinoma (A, ×50; B and C, ×160). Strong cytoplasmic labeling isdetected in the stromal cells, in contrast to the neoplastic epitheliumthat is negative for SPARC.

FIG. 3 represents (a) Distribution of CpG dinucleotides (vertical lines)in the 5′ region of the SPARC gene showing a CpG-rich sequence (CpGisland) spanning from exon 1 to intron 1; (b) Methylation-specific PCR(MSP) analysis of SPARC in pancreatic cancer cell lines and anon-neoplastic HPDE; the PCR products in the lanes U and M indicate thepresence of umethylated and methylated templates, respectively; (c)SPARC mRNA expression by RT-PCR in pancreatic cancer cell linesharboring aberrant SPARC methylation before (−) and after (+) treatmentwith 5-aza-2′-deoxycytidie (5Aza-dC); (d) MSP analysis of SPARC inpancreatic cancer xenografts; (e) MSP analysis of SPARC in normalpancreatic ductal epithelia selectively microdissected.

FIG. 4 represents the effects of exogenous SPARC on proliferation ofpancreatic cancer cells in vitro; two pancreatic cancer cell lines(AsCP1 and Panc1) were treated with or without SPARC (10 μg/ml), andcell number was counted 72 hours after treatment; the cell numbers shownare the means±SD of six measurements from three independent wells.

FIG. 5 represents (a) Semiquantitative RT-PCR analysis of SPARCexpression in primary fibroblasts derived from chronic pancreatitistissue (panc-f1), from non-cancerous pancreatic tissue from a patientwith pancreatic cancer (panc-f3), and from pancreatic adenocarcinomatissue (panc-f5); the bar graph shown represents relative SPARC mRNAexpression for each sample normalized to the corresponding GAPDHexpression; (b) Change in SPARC mRNA expression in fibroblasts (panc-f3)upon co-culture with pancreatic cancer cells (CFPAC1); the bar graphrepresents the mean±SD of relative SPARC expression levels (normalizedto GAPDH) from two independent PCR reactions; (c) Effect of TGF-β onSPARC mRNA expression in fibroblasts (panc-f3); the bar graph representsthe mean±SD of relative SPARC expression levels (normalized to GAPDH)from two independent PCR reactions.

FIG. 6 represents the nucleic acid sequence for the human SPARC gene(SEQ ID NO: 1); Accession Number X82259.

FIG. 7 represents the nucleic acid sequence for the bisulfite sequencingprimers; forward (SEQ ID NO: 2) and reverse (SEQ ID NO: 3).

FIG. 8 represents the methylation specific PCR primers: Umethylated,forward (SEQ ID NO: 4) and reverse (SEQ ID NO: 5); and Methylated,forward (SEQ ID NO: 6) and reverse (SEQ ID NO: 7).

DETAILED DESCRIPTION OF THE INVENTION

It is understood that this invention is not limited to the particularmaterials and methods described herein. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. As usedherein, the singular forms “a”, “an”, and “the” include plural referenceunless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., Dictionary of Microbiology andMolecular Biology (2nd ed. 1994); The Cambridge Dictionary of Scienceand Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

All publications mentioned herein are cited for the purpose ofdescribing and disclosing the cell lines, protocols, reagents andvectors which are reported in the publications and which might be usedin connection with the invention. Nothing herein is to be construed asan admission that the invention is not entitled to antedate suchdisclosure by virtue of prior invention.

Definitions

“Biomarker” in the context of the present invention refers to a nucleicacid molecule which is present in a sample taken from patients havinghuman cancer as compared to a comparable sample taken from controlsubjects (e.g., a person with a negative diagnosis or undetectablecancer, normal or healthy subject). In the context of the presentinvention, the biomarker is specifically methylated SPARC, as identifiedin SEQ ID NO: 1 or a variant thereof.

“Diagnostic” means identifying the presence or nature of a pathologiccondition. In the context of the present invention with regard tocancer, the presense of a methylated SPARC nucleic acid is diagnostic ofcancer, and in particular pancreatic cancer, Diagnostic methods differin their sensitivity and specificity. The “sensitivity” of a diagnosticassay is the percentage of diseased individuals who test positive(percent of “true positives”). Diseased individuals not detected by theassay are “false negatives.” Subjects who are not diseased and who testnegative in the assay, are termed “true negatives.” The “specificity” ofa diagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

A “test amount” of a marker refers to an amount of a marker present in asample being tested. A test amount can be either in absolute amount(e.g., μg/ml) or a relative amount (e.g., relative intensity ofsignals).

A “diagnostic amount” of a marker refers to an amount of a marker in asubject's sample that is consistent with a diagnosis of human cancer. Adiagnostic amount can be either in absolute amount (e.g., μg/ml) or arelative amount (e.g., relative intensity of signals).

A “control amount” of a marker can be any amount or a range of amountwhich is to be compared against a test amount of a marker. For example,a control amount of a marker can be the amount of a marker in a personwithout human cancer. A control amount can be either in absolute amount(e.g., μg/ml) or a relative amount (e.g., relative intensity ofsignals).

“Detect” refers to identifying the presence, absence or amount of theobject to be detected.

By “patient” herein is meant a mammalian subject to be treated, withhuman patients being preferred. In some cases, the methods of theinvention find use in experimental animals, in veterinary application,and in the development of animal models for disease, including, but notlimited to, rodents including mice, rats, and hamsters; and primates.

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example, an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, or to shrink the cancer orprevent metastasis. The specific safe and effective amount ortherapeutically effective amount will vary with such factors as theparticular condition being treated, the physical condition of thepatient, the type of mammal or animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives.

As used herein, “proliferative growth disorder, “neoplastic disease,”“tumor; “cancer” are used interchangeably as used herein refers to acondition characterized by uncontrolled, abnormal growth of cells.Preferably the cancer to be treated is pancreatic cancer and theabnormal proliferation of cells in the pancreas can be any cell in theorgan. Examples of cancer include but are not limited to, carcinoma,blastoma, and sarcoma. As used herein, the term “carcinoma” refers to anew growth that arises from epithelium, found in skin or, more commonly,the lining of body organs.

The term “in need of such treatment” as used herein refers to a judgmentmade by a care giver such as a physician, nurse, or nurse practitionerin the case of humans that a patient requires or would benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a care giver's expertise, but that include the knowledgethat the patient is ill, or will be ill, as the result of a conditionthat is treatable by the compounds of the invention.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. “Treatment” may also bespecified as palliative care. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented. In tumor (e.g., cancer) treatment, a therapeutic agent maydirectly decrease the pathology of tumor cells, or render the tumorcells more susceptible to treatment by other therapeutic agents, e.g.,radiation and/or chemotherapy.

An “effective amount” of a composition disclosed herein or an agonistthereof, in reference to “inhibiting the cellular proliferation” of aneoplastic cell, is an amount capable of inhibiting, to some extent, thegrowth of target cells. The term further includes an amount capable ofinvoling a growth inhibitory, cytostatic and/or cytotoxic effect and/orapoptosis and/or necrosis of the target cells. An “effective amount” of,for example a potential candidate agent that interacts with the nucleicacid molecules described herein, for purposes of inhibiting neoplasticcell growth may be determined empirically and in a routine manner usingmethods well known in the art.

A “therapeutically effective amount”, in reference to the treatment ofneoplastic disease or neoplastic cells, refers to an amount capable ofinvoking one or more of the following effects: (1) inhibition, to someextent, of tumor growth, including, (i) slowing down and (ii) completegrowth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion or (v) reducing, slowing or preventing metastasis; and/or (8)relief, to some extent, of one or more symptoms associated with thedisorder.

In another aspect, the invention provides methods for detectingbiomarkers (i.e., methylated SPARC) which are present in the samples ofa human cancer patient and a control (e.g., an individual in whom humancancer is undetectable). The biomarkers can be detected in a number ofbiological samples. The sample is preferably a biological fluid, tissueor organ sample. Examples of a biological fluid sample useful in thisinvention include blood, blood serum, plasma, pancreatic fluids,aspirate, urine, tears, saliva, etc.

Detection of SPARC Nucleic Acid Molecules

The normal pancreas contains a predominance of acinar cells and isletsrelative to normal duct epithelium. The normal pancreatic ductepithelium is therefore underrepresented in gene expression analyses ofbulk normal pancreas. Therefore, in a preferred embodiment, the SPARCgene identified by a biochip, such as for example, Affymetrix GeneChip,are further refined to exclude genes highly expressed in cultures ofnormal pancreatic ductal epithelial cells. For each gene identified asdifferentially expressed by Affymetrix GeneChip, the corresponding SAGEtag was identified, and the total number of SAGE tags present in theSAGEmap database (http://www.ncbi.nlm.nih.gov/SAGE/) of normal pancreasduct epithelium libraries HX and H126 was determined. Preferably, anygene having at least about five tags in about one of these two SAGElibraries was then excluded from further analysis.

Serial Analysis of Gene Expression (SAGE), is based on theidentification of and characterization of partial, defined sequences oftranscripts corresponding to gene segments. These defined transcriptsequence “tags” are markers for genes which are expressed in a cell, atissue, or an extract, for example.

SAGE is based on several principles. First, a short nucleotide sequencetag (9 to 10 bp) contains sufficient information content to uniquelyidentify a transcript provided it is isolated from a defined positionwithin the transcript. For example, a sequence as short as 9 bp candistinguish 262,144 transcripts (4.sup.9) given a random nucleotidedistribution at the tag site, whereas estimates suggest that the humangenome encodes about 80,000 to 200,000 transcripts (Fields, et al.,Nature Genetics, 7:345 1994). The size of the tag can be shorter forlower eukaryotes or prokaryotes, for example, where the number oftranscripts encoded by the genome is lower. For example, a tag as shortas 6-7 bp may be sufficient for distinguishing transcripts in yeast.

Second, random dimerization of tags allows a procedure for reducing bias(caused by amplification and/or cloning). Third, concatenation of theseshort sequence tags allows the efficient analysis of transcripts in aserial manner by sequencing multiple tags within a single vector orclone. As with serial communication by computers, wherein information istransmitted as a continuous string of data, serial analysis of thesequence tags requires a means to establish the register and boundariesof each tag. The concept of deriving a defined tag from a sequence inaccordance with the present invention is useful in matching tags ofsamples to a sequence database. In the preferred embodiment, a computermethod is used to match a sample sequence with known sequences.

The tags used herein, uniquely identify genes. This is due to theirlength, and their specific location (3′) in a gene from which they aredrawn. The full length genes can be identified by matching the tag to agene data base member, or by using the tag sequences as probes tophysically isolate previously unidentified genes from cDNA libraries.The methods by which genes are isolated from libraries using DNA probesare well known in the art. See, for example, Veculescu et al., Science270: 484 (1995), and Sambrook et al. (1989), MOLECULAR CLONING: ALABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Once a gene or transcript has been identified, either bymatching to a data base entry, or by physically hybridizing to a cDNAmolecule, the position of the hybridizing or matching region in thetranscript can be determined. If the tag sequence is not in the 3′ end,immediately adjacent to the restriction enzyme used to generate the SAGEtags, then a spurious match may have been made. Confirmation of theidentity of a SAGE tag can be made by comparing transcription levels ofthe tag to that of the identified gene in certain cell types.

Analysis of gene expression is not limited to the above method but caninclude any method known in the art. All of these principles may beapplied independently, in combination, or in combination with otherknown methods of sequence identification.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutchiffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF)(Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

In a preferred embodiment, Expressed Sequenced Tags (ESTs), can also beused to identify nucleic acid molecules which are over expressed in acancer cell. ESTs from a variety of databases can be indentified. Forexample, preferred databases include, for example, Online MendelianInheritance in Man (OMIM), the Cancer Genome Anatomy Project (CGAP),GenBank, EMBL, PIR, SWISS-PROT, and the like. OMIM, which is a databaseof genetic mutations associated with disease, was developed, in part,for the National Center for Biotechnology Information (NCBI). OMIM canbe accessed through the world wide web of the Internet, at, for example,ncbi.nlm.nih.gov/Omim/. CGAP, which is an interdisciplinary program toestablish the information and technological tools required to decipherthe molecular anatomy of a cancer cell. CGAP can be accessed through theworld wide web of the Internet, at, for example,ncbi.nlm.nih.gov/ncicgap/. Some of these databases may contain completeor partial nucleotide sequences. In addition, alternative transcriptforms can also be selected from private genetic databases.Alternatively, nucleic acid molecules can be selected from availablepublications or can be determined especially for use in connection withthe present invention.

Alternative transcript forms can be generated from individual ESTs whichare within each of the databases by computer software which generatescontiguous sequences. In another embodiment of the present invention,the nucleotide sequence of the nucleic acid molecule is determined byassembling a plurality of overlapping ESTs. The EST database (dbEST),which is known and available to those skilled in the art, comprisesapproximately one million different human mRNA sequences comprising fromabout 500 to 1000 nucleotides, and various numbers of ESTs from a numberof different organisms. dbEST can be accessed through the world wide webof the Internet, at, for example, ncbi.nlm.nih.gov/dbEST/index.html.These sequences are derived from a cloning strategy that uses cDNAexpression clones for genome sequencing. ESTs have applications in thediscovery of new genes, mapping of genomes, and identification of codingregions in genomic sequences. Another important feature of EST sequenceinformation that is becoming rapidly available is tissue-specific geneexpression data. This can be extremely useful in targeting selectivegene(s) for therapeutic intervention. Since EST sequences are relativelyshort, they must be assembled in order to provide a complete sequence.Because every available clone is sequenced, it results in a number ofoverlapping regions being reported in the database. The end result isthe elicitation of alternative transcript forms from, for example,normal cells and cancer cells.

Assembly of overlapping ESTs extended along both the 5′ and 3′directions results in a full-length “virtual transcript.” The resultantvirtual transcript may represent an already characterized nucleic acidor may be a novel nucleic acid with no known biological function. TheInstitute for Genomic Research (TIGR) Human Genome Index (HGI) database,which is known and available to those skilled in the art, contains alist of human transcripts. TIGR can be accessed through the world wideweb of the Internet, at, for example, tigr.org. Transcripts can begenerated in this manner using TIGR-Assembler, an engine to buildvirtual transcripts and which is known and available to those skilled inthe art. TIGR-Assembler is a tool for assembling large sets ofoverlapping sequence data such as ESTs, BACs, or small genomes, and canbe used to assemble eukaryotic or prokaryotic sequences. TIGR-Assembleris described in, for example, Sutton, et al., Genome Science & Tech.,1995, 1, 9-19, which is incorporated herein by reference in itsentirety, and can be accessed through the file transfer program of theInternet, at, for example, tigr.org/pub/software/TIGR. assembler. Inaddition, GLAXO-MRC, which is known and available to those skilled inthe art, is another protocol for constructing virtual transcripts. Inaddition, “Find Neighbors and Assemble EST Blast” protocol, which runson a UNIX platform, has been developed by Applicants to constructvirtual transcripts. PHRAP is used for sequence assembly within FindNeighbors and Assemble EST Blast. PHRAP can be accessed through theworld wide web of the Internet, at, for example,chimera.biotech.washington.edu/uwgc/tools/phrap.htm. Identification ofESTs and generation of contiguous ESTs to form full length RNA moleculesis described in detail in U.S. application Ser. No. 09/076,440, which isincorporated herein by reference in its entirety.

In yet another aspect, variants of the nucleic acid molecules asidentified in FIGS. 1A through 1M can be used to detect pancreaticcancers. An “allele” or “variant” is an alternative form of a gene. Ofparticular utility in the invention are variants of the genes encodingany potential pancreatic tumor markers identified by the methods of thisinvention. Variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes that give rise to variants are generally ascribed tonatural deletions, additions, or substitutions of nucleotides. Each ofthese types of changes may occur alone, or in combination with theothers, one or more times in a given sequence.

To further identify variant nucleic acid molecules which can detect, forexample, pancreatic cancer at an early stage, nucleic acid molecules canbe grouped into sets depending on the homology, for example. The membersof a set of nucleic acid molecules are compared. Preferably, the set ofnucleic acid molecules is a set of alternative transcript forms ofnucleic acid. Preferably, the members of the set of alternativetranscript forms of nucleic acids include at least one member which isassociated, or whose encoded protein is associated, with a disease stateor biological condition. Thus, comparison of the members of the set ofnucleic acid molecules results in the identification of at least onealternative transcript form of nucleic acid molecule which isassociated, or whose encoded protein is associated, with a disease stateor biological condition. In a preferred embodiment of the invention, themembers of the set of nucleic acid molecules are from a common gene. Inanother embodiment of the invention, the members of the set of nucleicacid molecules are from a plurality of genes. In another embodiment ofthe invention, the members of the set of nucleic acid molecules are fromdifferent taxonomic species. Nucleotide sequences of a plurality ofnucleic acids from different taxonomic species can be identified byperforming a sequence similarity search, an ortholog search, or both,such searches being known to persons of ordinary skill in the art.

Sequence similarity searches can be performed manually or by usingseveral available computer programs known to those skilled in the art.Preferably, Blast and Smith-Waterman algorithms, which are available andknown to those skilled in the art, and the like can be used. Blast isNCBI's sequence similarity search tool designed to support analysis ofnucleotide and protein sequence databases. Blast can be accessed throughthe world wide web of the Internet, at, for example,ncbi.nln.nih.gov/BLAST/. The GCG Package provides a local version ofBlast that can be used either with public domain databases or with anylocally available searchable database. GCG Package v9.0 is acommercially available software package that contains over 100interrelated software programs that enables analysis of sequences byediting, mapping, comparing and aligning them. Other programs includedin the GCG Package include, for example, programs which facilitate RNAsecondary structure predictions, nucleic acid fragment assembly, andevolutionary analysis. In addition, the most prominent genetic databases(GenBank, EMBL, PIR, and SWISS-PROT) are distributed along with the GCGPackage and are fully accessible with the database searching andmanipulation programs. GCG can be accessed through the Internet at, forexample, http://www.gcg.com/. Fetch is a tool available in GCG that canget annotated GenBank records based on accession numbers and is similarto Entrez. Another sequence similarity search can be performed withGeneWorld and GeneThesaurus from Pangea. GeneWorld 2.5 is an automated,flexible, high-throughput application for analysis of polynucleotide andprotein sequences. GeneWorld allows for automatic analysis andannotations of sequences. Like GCG, GeneWorld incorporates several toolsfor homology searching, gene finding, multiple sequence alignment,secondary structure prediction, and motif identification. GeneThesaurus1.0 tm is a sequence and annotation data subscription service providinginformation from multiple sources, providing a relational data model forpublic and local data.

Another alternative sequence similarity search can be performed, forexample, by BlastParse. BlastParse is a PERL script running on a UNIXplatform that automates the strategy described above. BlastParse takes alist of target accession numbers of interest and parses all the GenBankfields into “tab-delimited” text that can then be saved in a “relationaldatabase” format for easier search and analysis, which providesflexibility. The end result is a series of completely parsed GenBankrecords that can be easily sorted, filtered, and queried against, aswell as an annotations-relational database.

Preferably, the plurality of nucleic acids from different taxonomicspecies which have homology to the target nucleic acid, as describedabove in the sequence similarity search, are further delineated so as tofind orthologs of the target nucleic acid therein. An ortholog is a termdefined in gene classification to refer to two genes in widely divergentorganisms that have sequence similarity, and perform similar functionswithin the context of the organism. In contrast, paralogs are geneswithin a species that occur due to gene duplication, but have evolvednew functions, and are also referred to as isotypes. Optionally, paralogsearches can also be performed. By performing an ortholog search, anexhaustive list of homologous sequences from as diverse organisms aspossible is obtained. Subsequently, these sequences are analyzed toselect the best representative sequence that fits the criteria for beingan ortholog. An ortholog search can be performed by programs availableto those skilled in the art including, for example, Compare. Preferably,an ortholog search is performed with access to complete and parsedGenBank annotations for each of the sequences. Currently, the recordsobtained from GenBank are “flat-files”, and are not ideally suited forautomated analysis. Preferably, the ortholog search is performed using aQ-Compare program. Preferred steps of the Q-Compare protocol aredescribed in the flowchart set forth in U.S. Pat. No. 6,221,587,incorporated herein by reference.

Preferably, interspecies sequence comparison is performed using Compare,which is available and known to those skilled in the art. Compare is aGCG tool that allows pair-wise comparisons of sequences using awindow/stringency criterion. Compare produces an output file containingpoints where matches of specified quality are found. These can beplotted with another GCG tool, DotPlot.

The SPARC nucleic acid molecules of this invention can be isolated usingthe technique described in the experimental section or replicated usingPCR. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065, and 4,683,202 and described in PCR: ThePolymerase Chain Reaction (Mullis et al. eds, Birkhauser Press, Boston(1994)) or MacPherson et al. (1991) and (1994), supra, and referencescited therein (see Methylation Specific PCR below). Alternatively, oneof skill in the art can use the sequences provided herein and acommercial DNA synthesizer to replicate the DNA. Accordingly, thisinvention also provides a process for obtaining the polynucleotides ofthis invention by providing the linear sequence of the polynucleotide,nucleotides, appropriate primer molecules, chemicals such as enzymes andinstructions for their replication and chemically replicating or linkingthe nucleotides in the proper orientation to obtain the polynucleotides.In a separate embodiment, these polynucleotides are further isolated.Still further, one of skill in the art can insert the polynucleotideinto a suitable replication vector and insert the vector into a suitablehost cell (procaryotic or eucaryotic) for replication and amplification.The DNA so amplified can be isolated from the cell by methods well knownto those of skill in the art. A process for obtaining polynucleotides bythis method is further provided herein as well as the polynucleotides soobtained.

The terms “nucleic acid molecule” and “tumor marker” or “polynucleotide”will be used interchangeably throughout the specification, unlessotherwise specified. As used herein, “nucleic acid molecule” refers tothe phosphate ester polymeric form of ribonucleosides (adenosine,guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester analogues thereof, such asphosphorothioates and thioesters, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter alia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of DNA (i.e., the strandhaving a sequence homologous to the mRNA). A “recombinant DNA molecule”is a DNA molecule that has undergone a molecular biologicalmanipulation.

In an embodiment of the invention the presence of amethylated SPARCnucleic acid molecule is correlated to a sample of a normal subject. Thesample is preferably obtained from a mammal suspected of having aproliferative cell growth disorder, in particular, a pancreatic cancer.Preferably, a nucleic acid molecule that is indicative of a cancercomprises a sequence having at least about 80% sequence identity to amolecule identified in SEQ ID NO: 1, more preferably the nucleic acidmolecule comprises a sequence having at least about 90% sequenceidentity to a molecule identified in SEQ ID NO: 1, most preferably thenucleic acid molecule comprises a sequence having at least about 95%sequence identity to a molecule identified in SEQ ID NO: 1.

In another preferred embodiment, the nucleic acid molecule is expressedat a lower level in a patient with cancer as compared to expressionlevels in a normal individual. Preferably the nucleic acid molecule isexpressed at least about 15 fold lower in a patient with cancer ascompared to expression in a normal individual, more preferably thenucleic acid molecule is expressed at least about 10 fold lower in apatient with cancer as compared to expression in a normal individual,most preferably the nucleic acid molecule is expressed at least about 5fold lower in a patient with cancer as compared to expression in anormal individual.

Percent identity and similarity between two sequences (nucleic acid orpolypeptide) can be determined using a mathematical algorithm (see,e.g., Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps are introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap which need to beintroduced for optimal alignment of the two sequences. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions, respectively, are then compared. When a positionin the first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”).

A “comparison window” refers to a segment of any one of the number ofcontiguous positions selected from the group consisting of from 25 to600, usually about 50 to about 200, more usually about 100 to about 150in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well-knownin the art.

For example, the percent identity between two amino acid sequences canbe determined using the Needleman and Wunsch algorithm (J. Mol. Biol.(48): 444-453, 1970) which is part of the GAP program in the GCGsoftware package (available at http://www.gcg.com), by the localhomology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482, 1981),by the search for similarity methods of Pearson & Lipman (Proc. Natl.Acad. Sci. USA 85: 2444, 1988) and Altschul, et al. (Nucleic Acids Res.25(17): 3389-3402, 1997), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and BLAST in the Wisconsin GeneticsSoftware Package (available from, Genetics Computer Group, 575 ScienceDr., Madison, Wis.), or by manual alignment and visual inspection (see,e.g., Ausubel et al., supra). Gap parameters can be modified to suit auser's needs. For example, when employing the GCG software package, aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6 can be used. Examplary gap weightsusing a Blossom 62 matrix or a PAM250 matrix, are 16, 14, 12, 10, 8, 6,or 4, while exemplary length weights are 1, 2, 3, 4, 5, or 6. The GCGsoftware package can be used to determine percent identity betweennucleic acid sequences. The percent identity between two amino acid ornucleotide sequences also can be determined using the algorithm of E.Myers and W. Miller (CABIOS 4: 11-17, 1989) which has been incorporatedinto the ALIGN program (version 2.0), using a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid sequences of the present invention can further be usedas query sequences to perform a search against sequence databases to,for example, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (J. Mol. Biol. 215: 403-10, 1990). BLASTnucleotide searches can be performed with the NBLAST program, withexemplary scores=100, and wordlengths=12 to obtain nucleotide sequenceshomologous to or with sufficient percent identity to the nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, with exemplary scores=50 and wordlengths=3 to obtainamino acid sequences sufficiently homologous to or with sufficient %identity to the proteins of the invention. To obtain gapped alignmentsfor comparison purposes, gapped BLAST can be used as described inAltschul et al. (Nucleic Acids Res. 25(17): 3389-3402, 1997). When usingBLAST and gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual Second Edition (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook etal., 1989”); DNA Cloning: A Practical Approach. Volumes I and II (D. N.Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];Transcription And Translation [B. D. Hames & S. J. Higgins, eds.(1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide ToMolecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. (1994).

As used herein, the term “fragment or segment”, as applied to a nucleicacid sequence, gene, will ordinarily be at least about 5 contiguousnucleic acid bases (for nucleic acid sequence or gene) or amino acids(for polypeptides), typically at least about 10 contiguous nucleic acidbases or amino acids, more typically at least about 20 contiguousnucleic acid bases or amino acids, usually at least about 30 contiguousnucleic acid bases or amino acids, preferably at least about 40contiguous nucleic acid bases or amino acids, more preferably at leastabout 50 contiguous nucleic acid bases or amino acids, and even morepreferably at least about 60 to 80 or more contiguous nucleic acid basesor amino acids in length. “Overlapping fragments” as used herein, referto contiguous nucleic acid fragments which begin at the amino terminalend of a nucleic acid and end at the carboxy terminal end of the nucleicacid or protein. Each nucleic acid or fragment has at least about onecontiguous nucleic acid position in common with the next nucleic acidfragment, more preferably at least about three contiguous nucleic acidbases in common, most preferably at least about ten contiguous nucleicacid bases in common.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least 20nucleotides, more generally at least 23 nucleotides, ordinarily at least26 nucleotides, more ordinarily at least 29 nucleotides, often at least32 nucleotides, more often at least 35 nucleotides, typically at least38 nucleotides, more typically at least 41 nucleotides, usually at least44 nucleotides, more usually at least 47 nucleotides, preferably atleast 50 nucleotides, more preferably at least 53 nucleotides, and inparticularly preferred embodiments will be at least 56 or morenucleotides. Additional preferred embodiments will include lengths inexcess of those numbers, e.g., 63, 72, 87, 96, 105, 117, etc. Saidfragments may have termini at any pairs of locations, but especially atboundaries between structural domains, e.g., membrane spanning portions.

Homologous nucleic acid sequences, when compared, exhibit significantsequence identity or similarity. The standards for homology in nucleicacids are either measures for homology generally used in the art bysequence comparison or based upon hybridization conditions. Thehybridization conditions are described in greater detail below.

As used herein, “substantial homology” in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least 56%, moregenerally at least 59%, ordinarily at least 62%, more ordinarily atleast 65%, often at least 68%, more often at least 71%, typically atleast 74%, more typically at least 77%, usually at least 80%, moreusually it least about 85%, preferably at least about 90%, morepreferably at least about 95 to 98% or more, and in particularembodiments, as high at about 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a fragment derived from FIGS. 1A through 1M,e.g., 39829_at. Typically, selective hybridization will occur when thereis at least about 55% homology over a stretch of at least about 14nucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. See, Kanehisa (1984)Nuc. Acids Res. 12:203-213. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willbe over a stretch of at least about 17 nucleotides, usually at leastabout 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides. The endpointsof the segments may be at many different pair combinations.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 1000 mM, usually less than about 500 mM,more usually less than about 400 mM, typically less than about 300 mM,preferably less than about 200 mM, and more preferably less than about150 mM. However, the combination of parameters is much more importantthan the measure of any single parameter. See, e.g., Wetmur and Davidson(1968) J. Mol. Biol. 31:349-370.

Methylation Specific Polymerase Chain Reaction (MSP)

In one embodiment, the invention provides a method for detecting amethylated CpG-containing SPARC nucleic acid, the method includingcontacting a nucleic acid-containing specimen with an agent thatmodifies unmethylated cytosine; amplifying the CpG-containing nucleicacid in the specimen by means of CpG-specific oligonucleotide primers;and detecting the methylated nucleic acid. It is understood that whilethe amplification step is optional, it is desirable in the preferredmethod of the invention.

The term “modifies” as used herein means the conversion of anunmethylated cytosine to another nucleotide which will distinguish theunmethylated from the methylated cytosine. Preferably, the agentmodifies unmethylated cytosine to uracil. Preferably, the agent used formodifying unmethylated cytosine is sodium bisulfite, however, otheragents that similarly modify unmethylated cytosine, but not methylatedcytosine can also be used in the method of the invention. Sodiumbisulfite (NaHSO₃) reacts readily with the 5,6-double bond of cytosine,but poorly with methylated cytosine. Cytosine reacts with the bisulfiteion to form a sulfonated cytosine reaction intermediate which issusceptible to deamination, giving rise to a sulfonated uracil. Thesulfonate group can be removed under alkaline conditions, resulting inthe formation of uracil. Uracil is recognized as a thymine by Taqpolymerase and therefore upon PCR, the resultant product containscytosine only at the position where 5-methylcytosine occurs in thestarting template DNA.

The primers used in the invention for amplification of theCpG-containing nucleic acid in the specimen, after bisulfitemodification, specifically distinguish between untreated DNA,methylated, and non-methylated DNA. MSP primers for the non-methylatedDNA preferably have a T in the 3′ CG pair to distinguish it from the Cretained in methylated DNA, and the compliment is designed for theantisense primer. MSP primers usually contain relatively few Cs or Gs inthe sequence since the Cs will be absent in the sense primer and the Gsabsent in the antisense primer (C becomes modified to U (uracil) whichis amplified as T (thymidine) in the amplification product).

The primers of the invention embrace oligonucleotides of sufficientlength and appropriate sequence so as to provide specific initiation ofpolymerization on a significant number of nucleic acids in thepolymorphic locus. Specifically, the term “primer” as used herein refersto a sequence comprising two or more deoxyribonucleotides orribonucleotides, preferably more than three, and most preferably morethan 8, which sequence is capable of initiating synthesis of a primerextension product, which is substantially complementary to a polymorphiclocus strand. Environmental conditions conducive to synthesis includethe presence of nucleoside triphosphates and an agent forpolymerization, such as DNA polymerase, and a suitable temperature andpH. The primer is preferably single stranded for maximum efficiency inamplification, but may be double stranded. If double stranded, theprimer is first treated to separate its strands before being used toprepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the inducing agentfor polymerization. The exact length of primer will depend on manyfactors, including temperature, buffer, and nucleotide composition. Theoligonucleotide primer typically contains 12-20 or more nucleotides,although it may contain fewer nucleotides.

Primers of the invention are designed to be “substantially”complementary to each strand of the genomic locus to be amplified andinclude the appropriate G or C nucleotides as discussed above. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands under conditions which allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with the 5′ and 3′ flanking sequences tohybridize therewith and permit amplification of the genomic locus.

Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of target locus relative to the number ofreaction steps involved. Typically, one primer is complementary to thenegative (−) strand of the locus and the other is complementary to thepositive (+) strand. Annealing the primers to denatured nucleic acidfollowed by extension with an enzyme, such as the large fragment of DNAPolymerase I and nucleotides, results in newly synthesized + and −strands containing the target locus sequence. Because these newlysynthesized sequences are also templates, repeated cycles of denaturing,primer annealing, and extension results in exponential production of theregion (i.e., the target locus sequence) defined by the primer. Theproduct of the chain reaction is a discrete nucleic acid duplex withtermini corresponding to the ends of the specific primers employed.

The oligonucleotide primers of the invention may be prepared using anysuitable method, such as conventional phosphotriester and phosphodiestermethods or automated embodiments thereof. In one such automatedembodiment, diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage, et al. (TetrahedronLetters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

Any nucleic acid specimen, in purified or nonpurified form, can beutilized as the starting nucleic acid or acids, provided it contains, oris suspected of containing, the specific nucleic acid sequencecontaining the target locus (e.g., CpG). Thus, the process may employ,for example, DNA or RNA, including messenger RNA, wherein DNA or RNA maybe single stranded or double stranded. In the event that RNA is to beused as a template, enzymes, and/or conditions optimal for reversetranscribing the template to DNA would be utilized. In addition, aDNA-RNA hybrid which contains one strand of each may be utilized. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so utilized. The specific nucleic acid sequenceto be amplified, i.e., the target locus, may be a fraction of a largermolecule or can be present initially as a discrete molecule, so that thespecific sequence constitutes the entire nucleic acid. It is notnecessary that the sequence to be amplified be present initially in apure form; it may be a minor fraction of a complex mixture, such ascontained in whole human DNA.

The nucleic acid-containing specimen used for detection of methylatedCpG may be from any source including brain, colon, urogenital,hematopoietic, thymus, testis, ovarian, uterine, prostate, breast,colon, lung and renal tissue and may be extracted by a variety oftechniques such as that described by Maniatis, et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp 280, 281,1982).

If the extracted sample is impure (such as plasma, serum, or blood or asample embedded in parrafin), it may be treated before amplificationwith an amount of a reagent effective to open the cells, fluids,tissues, or animal cell membranes of the sample, and to expose and/orseparate the strand(s) of the nucleic acid(s). This lysing and nucleicacid denaturing step to expose and separate the strands will allowamplification to occur much more readily.

Where the target nucleic acid sequence of the sample contains twostrands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means, the word “denaturing” includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80.degree. to105.degree. C. for times ranging from about 1 to 10 minutes. Strandseparation may also be induced by an enzyme from the class of enzymesknown as helicases or by the enzyme RecA, which has helicase activity,and in the presence of riboATP, is known to denature DNA. The reactionconditions suitable for strand separation of nucleic acids withhelicases are described by Kuhn Hoffmann-Berling (CSH-QuantitativeBiology, 43:63, 1978) and techniques for using RecA are reviewed in C.Radding (Ann. Rev. Genetics, 16:405-437, 1982).

When complementary strands of nucleic acid or acids are separated,regardless of whether the nucleic acid was originally double or singlestranded, the separated strands are ready to be used as a template forthe synthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates to occur. Generally synthesis occurs in a buffered aqueoussolution, preferably at a pH of 7-9, most preferably about 8.Preferably, a molar excess (for genomic nucleic acid, usually about10.sup.8:1 primer:template) of the two oligonucleotide primers is addedto the buffer containing the separated template strands. It isunderstood, however, that the amount of complementary strand may not beknown if the process of the invention is used for diagnosticapplications, so that the amount of primer relative to the amount ofcomplementary strand cannot be determined with certainty. As a practicalmatter, however, the amount of primer added will generally be in molarexcess over the amount of complementary strand (template) when thesequence to be amplified is contained in a mixture of complicatedlong-chain nucleic acid strands. A large molar excess is preferred toimprove the efficiency of the process.

The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP areadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90.degree.-100.degree. C. from about 1 to 10 minutes, preferablyfrom 1 to 4 minutes. After this heating period, the solution is allowedto cool to room temperature, which is preferable for the primerhybridization. To the cooled mixture is added an appropriate agent foreffecting the primer extension reaction (called herein “agent forpolymerization”), and the reaction is allowed to occur under conditionsknown in the art. The agent for polymerization may also be addedtogether with the other reagents if it is heat stable. This synthesis(or amplification) reaction may occur at room temperature up to atemperature above which the agent for polymerization no longerfunctions. Thus, for example, if DNA polymerase is used as the agent,the temperature is generally no greater than about 40.degree. C. Mostconveniently the reaction occurs at room temperature.

The agent for polymerization may be any compound or system which willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productswhich are complementary to each locus nucleic acid strand. Generally,the synthesis will be initiated at the 3′ end of each primer and proceedin the 5′ direction along the template strand, until synthesisterminates, producing molecules of different lengths. There may beagents for polymerization, however, which initiate synthesis at the 5′end and proceed in the other direction, using the same process asdescribed above.

Preferably, the method of amplifying is by PCR, as described herein andas is commonly used by those of ordinary skill in the art. Alternativemethods of amplification have been described and can also be employed aslong as the methylated and non-methylated loci amplified by PCR usingthe primers of the invention is similarly amplified by the alternativemeans.

The amplified products are preferably identified as methylated ornon-methylated by sequencing. Sequences amplified by the methods of theinvention can be further evaluated, detected, cloned, sequenced, and thelike, either in solution or after binding to a solid support, by anymethod usually applied to the detection of a specific DNA sequence suchas PCR, oligomer restriction (Saiki, et al., Bio/Technology,3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis(Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278, 1983),oligonucleotide ligation assays (OLAs) (Landegren, et al., Science,241:1077, 1988), and the like. Molecular techniques for DNA analysishave been reviewed (Landegren, et al., Science, 242:229-237, 1988

Optionally, the methylation pattern of the nucleic acid can be confirmedby restriction enzyme digestion and Southern blot analysis. Examples ofmethylation sensitive restriction endonucleases which can be used todetect 5′CpG methylation include SmaI, SacII, EagI, MspI, HpaII, BstUIand BssHII, for example.

Treatment of Methylated Sparc Gene Related Cancers

DNMT inhibitors, such as 5-aza-cytidine (5-aza-CR) and5-aza-2′-deoxycytidine (5-aza-CdR) are also widely studied because DNAhypomethylation induces the re-activation of tumor suppressor genes thatare silenced by methylation-mediated mechanisms, and in particular, themethylated SPARC gene. The combination of HDAC inhibitors ordemethylating agents with other chemo-therapeutics can be used as apossible molecularly targeted therapeutic strategy. In particular, thecombination of HDAC inhibitors with demethylating agents are effectivesince histones are connected to DNA by both physical and functionalinteractions. As such, the combination of HDAC and DNMT inhibition canbe very effective (and synergistic) in inducing apoptosis,differentiation and/or cell growth arrest in human pancreatic lung,breast, thoracic, leukemia and colon cancer cell lines. Effective agentsinclude HDAC inhibitors, such as trichostatin A (TSA), sodium butyrate,depsipeptide (FR901228, FK228), valproic acid (VPA) and suberoylanilidehydroxamic acid (SAHA), and the demethylating agent, 5-aza-CdR usedalone and in combination treatment of human cancer cells.

Diagnostic Kits

In another aspect, the invention provides kits for diagnosis of humancancer, wherein the kits can be used to detect the biomarker of thepresent invention. For example, the kits can be used to detect themethylated SPARC nucleic acid described herein, which biomarker ispresent in samples of a human cancer patient and not in normal subjects.The kits of the invention have many applications. For example, the kitscan be used to differentiate if a subject has human cancer or has anegative diagnosis, thus aiding a human cancer diagnosis. In anotherexample, the kits can be used to identify compounds that modulateexpression of the biomarker in in vitro or in vivo animal models forhuman cancer.

Optionally, the kit may further comprise a standard or controlinformation so that the test sample can be compared with the controlinformation standard to determine if the test-amount of a biomarkerdetected in a sample is a diagnostic amount consistent with a diagnosisof human cancer.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

EXAMPLES

Materials and Methods

Materials

Amonoclonal anti-SPARC antibody (clone ON1-1) was purchased from ZymedLaboratories, Inc. (South San Francisco, Calif.). 5-Aza-2′-deoxycytidine(5Aza-dC) and human recombinant transforming growth factor (TGF)-β1 werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Purified humanplatelet SPARC protein was purchased from Calbiochem (Cambridge, Mass.).

Cell Lines and Tissue Samples

Seventeen human pancreatic cancer cell lines (AsPC1, BxPC3, Capan1,Capan2, CFPAC1, Colo357, Hs766T, MiaPaCa2, Panc1, PL1, PL3, PL6, PL9,PL10, PL11, PL12, and PL13) were maintained in RPMI 1640 (Invitrogen,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS),streptomycin, and penicillin at 37° C. in a humidified atmospherecontaining 5% CO₂. An immortal cell line derived from normal humanpancreatic ductal epithelium (HPDE) was generously provided by Dr.Ming-Sound Tsao (University of Toronto, Ontario) and maintained inKeratinocyte-SFM (Invitrogen). Primary fibroblasts were initiallyoutgrown from chronic pancreatitis tissue from a 33-year-old malepatient (panc-f1), from non-cancerous pancreatic tissue from a61-year-old female patient with pancreatic cancer (panc-f3), or frompancreatic adenocarcinoma tissue from a 55-year-old female patient(panc-f5). These fibroblast cultures were carefully evaluated by lightmicroscopy to exclude epithelial cell contamination, maintained in RPMI1640 with 10% FBS, and used at 5-10 passages. Formalin-fixedparaffin-embedded blocks of 25 primary pancreatic adenocarcinomasresected at The Johns Hopkins Hospital were selected on the basis oftissue availability. Pancreatic cancer xenografts were established fromsurgically resected primary pancreatic carcinomas (Hahn et al., 1995),and 24 xenografts were randomly selected for this study. Normalpancreatic duct epithelial cells were selectively microdissected fromresected pancreata from 10 patients (mean age, 64.3 years; range, 36-83)with various pancreatic disorders using a laser-capture microdissection(LCM) system. Serum samples from patients with pancreatic disease.

Oligonucleotide Array Hybridization and Data Analysis

Total RNA was isolated from cultured cells or frozen tissues usingTRIZOL reagent (Invitrogen, Carlsbad, Calif.). First- andsecond-stranded cDNA was synthesized from 10 μg of total RNA usingT7-(dT)₂₄ primer (Genset Corp., South La Jolla, Calif.) and SuperScriptChoice system (Invitrogen). Labeled cRNA was synthesized from thepurified cDNA by in vitro transcription (IVT) reaction using theBioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Inc.,Farmingdale, N.Y.) at 37° C. for 6 hours, and was purified using RNeasyMini Kit (QIAGEN, Valencia, Calif.). The cRNA was fragmented at 94° C.for 35 minutes in a fragmentation buffer (40 mmol/L Tris-acetate (pH8.1), 100 mmol/L potassium acetate, 30 mmol/L magnesium acetate). Thefragmented cRNA was then hybridized to the Human Genome U133A chips(Affymetrix, Santa Clara, Calif.) with 18,462 unique gene/ESTtranscripts at 45° C. for 16 hours. The washing and staining procedurewas performed in the Affymetrix Fluidics Station according to themanufacturer's instructions. The probes were then scanned using a laserscanner, and signal intensity for each transcript (background-subtractedand adjusted for noise) and detection call (present, absent, ormarginal) were determined using Microarray Suite Software 5.0(Affymetrix).

Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

Four μg of total RNA was reverse-transcribed using Superscript II(Invitrogen). The SPARC RT-PCR reaction was performed under thecondition as follow: 95° C. for 5 minutes; then 28 cycles of 95° C. for20 seconds, 63° C. for 20 seconds, and 72° C. for 20 seconds; and afinal extension of 4 minutes at 72° C. Primer sequences were 5′-AAG ATCCAT GAG AAT GAG AAG-3′ (forward) and 5′-AAA AGC GGG TGG TGC AAT G-3′(reverse). To check the integrity of mRNA, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was also amplified in the same PCR condition. Forsemiquantitative analysis, the RT-PCR was performed with primers forSPARC and GAPDH in duplex reactions, and range of linear amplificationfor both genes was examined with serial PCR cycles to determine theoptimal cycle. The relative intensity of SPARC mRNA expression was thencorrected for variable RNA recovery using the corresponding GAPDH mRNAmeasurement as a surrogate for total mRNA.

Immunohistochemistry

Five-μm sections were cut onto coated slides and deparaffinized byroutine techniques. Antigen retrieval was performed in 10 mM sodiumcitrate buffer (pH 6.0) heated at 95° C. in a steamer for 20 minutes.After blocking endogenous peroxidase activity with a 3% aqueous H₂O₂solution for 5 minutes, the sections were incubated with an anti-SPARCmonoclonal antibody at a final concentration of 4 μg/ml for 60 minutes.Labeling was detected with the Envision Plus Detection Kit (DAKO,Carpinteria, Calif.) following the protocol as suggested by themanufacturer, and all sections were counterstained with hematoxylin. Theextent of immunolabeling of SPARC was categorized into three groups: 0%,negative; = or <10%, focal; and >10%, positive. The intensity ofimmunolabeling was categorized as weak (+), moderate (+⁺), or strong(++⁺).

Methylation-Specific Polymerase Chain Reaction (MSP)

Methylation status of the SPARC gene was determined by MSP as describedpreviously (Herman et al., 1996). Briefly, 1 μg of genomic DNA wastreated with sodium bisulfite for 16 hours at 50° C. After purification,1 μl of the bisulfite-treated DNA was amplified using primers specificfor either the methylated or for the unmethylated DNA under theconditions as follows: 95° C. for 5 minutes; then 40 cycles of 95° C.for 20 seconds, 62° C. for 20 seconds, and 72° C. for 30 seconds; and afinal extension of 4 minutes at 72° C. Primer sequences were TTT TTT AGATTG TTT GGA GAG TG (forward) and AAC TAA CAA CAT AAA CAA AAA TAT C(reverse) for umethylated reactions (132 bp), and GAG AGC GCG TTT TOTTTG TC (forward) and AAC GAC GTAAAC GAAAAT ATC G (reverse) formethylated reactions (112 bp). Five μl of each PCR product were loadedonto 3% agarose gels and visualized by ethidium bromide staining.

5Aza-dC Treatment

Eight pancreatic cancer cell lines (AsPC1, BxPC3, Capan2, CFPAC1,Hs766T, MiaPaCa2, PL3, and PL12) were treated with 5Aza-dC. Cells in logphase growth were seeded in T-75 culture flasks. After overnightincubation, the cells were exposed continuously to 5Aza-dC (1 μM) for 4days, with a change of drug and culture medium every 24 hours.

SPARC Enzyme-Linked Immunosorbent Assay (ELISA)

Cells were seeded at a density of 1×10⁵ cells/well in 6-well plates.After overnight incubation, the cells were washed withphosphate-buffered saline (PBS) and incubated in 2 ml of serum-freemedium for 24 hours. The conditioned media were harvested andcentrifuged to remove cellular debris. SPARC concentration in theconditioned media was measured using an enzyme-linked immunosorbentassay (ELISA) kit (Haematological Technologies, Inc., Essex Junction,Vt.) according to the manufacturer's instructions. SPARC levels weremeasured in the serum of patients with pancreatic disease in similarfashion.

Treatment of Pancreatic Cancer Cells with SPARC

We treated two pancreatic cancer cell lines (AsPC1 and Panc1) withexogenous SPARC. Cells in log phase growth were seeded at a density of1×10⁴ cells/well in 24-well plates. After overnight incubation, cellswere treated with or without human platelet SPARC protein (10 μg/ml) for72 hours, and the number of cells were counted by hemacytometer in threeindependent wells.

Fibroblasts/Pancreatic Cancer Cells Co-Culture

Fibroblasts were seeded in 6-well plates and grown for 48-72 hours.Pancreatic cancer cells (CFPAC1) were then seeded into the upper chamberof a transwell apparatus (Becton Dickinson, Franklin Lakes, N.J.), whichphysically separated the tumor cells from the fibroblasts but allowedfor interaction between the cells via soluble factors. After 48-hourincubation, fibroblasts were washed with PBS and harvested bytrypsinization.

Statistical Analysis

Statistical analysis was performed using Fisher's exact probability testor unpaired Student's t test (two-tailed). Differences were consideredsignificant at P<0.05.

Example 1 Gene Expression Analysis of SPARC in Pancreatic Cancer bySerial Analysis of Gene Expression (SAGE) and OligonucleotideMicroarrays

Oligonucleotide microarrays have been used to identify genes that areinduced 5-fold or greater by treatment of pancreatic cancer cells with5Aza-dC (Sato et al., manuscript submitted). SPARC was one of the geneswe identified using this approach. We therefore analyzed the geneexpression and methylation status of the SPARC gene in pancreaticcancer. First, we searched an online SAGE database(http://www.ncbi.nlm.nih.gov/SAGE/) to determine the gene expressionpatterns of SPARC in short-term cultures of normal pancreatic ductalepithelium, pancreatic cancer cell lines, and primary pancreatic cancertissues. The SAGE Tag to Gene Mapping analysis showed that the Hs.111779tag (ATGTGAAGAG) corresponding to the SPARC gene was present in both oftwo libraries from normal pancreatic duct epithelial cell cultures (H126and HX), whereas the SPARC tag was not identified in 3 of 4 pancreaticcancer cell lines (FIG. 1A). By contrast, the SPARC tag was detected athigh levels in two primary pancreatic adenocarcinoma tissues (Panc91-16113 and Panc 96-6252), suggesting that this gene may be an“invasion-specific gene” a gene whose expression is specificallyidentified in tissue specimens of invasive pancreatic cancer but not inpassaged pancreatic cancer cell lines (Ryu et al., 2001).

We then determined the SPARC expression by analyzing global geneexpression profiling (U133A oligonucleotide microarrays, Affymetrix) intwo frozen tissue samples of normal pancreatic ductal epithelial cellsselectively microdissected by LCM, a non-neoplastic pancreaticepithelial cell line (HPDE), and 5 pancreatic cancer cell lines (AsPC1,CFPAC1, Hs766T, MiaPaCa2, and Panc1). The SPARC transcript was detectedin the normal pancreatic ductal epithelial cells and HPDE (FIG. 1B). Incontrast, SPARC was not expressed in 4 of the 5 pancreatic cancer celllines.

Example 2 Expression of SPARC mRNA in Pancreatic Cancer Cell Lines andPrimary Fibroblasts

RT-PCR was preformed to examine the expression of SPARC mRNA in a panelof 17 pancreatic cancer cell lines and in primary fibroblasts derivedfrom pancreatic adenocarcinoma tissue (panc-f5). The SPARC transcriptwas detectable in a non-neoplastic pancreatic ductal epithelial cellline (HPDE) and was strongly expressed in the pancreatic cancer-derivedfibroblasts, whereas the expression was absent in 15 (88%) of the 17pancreatic cancer cell lines (FIG. 1C). Of note, the RT-PCR results of 7pancreatic cancer cell lines (AsPC1, Capan1, Capan2, CFPAC1, Hs766T,MiaPaCa2, and Panc1) parallel the SAGE and/or oligonucleotide array dataon these same cell lines. These results demonstrate the strikingdifference in SPARC expression between most pancreatic cancer cell linesand stromal fibroblasts.

Example 3 Immunohistochemical Analysis of SPARC Expression in PancreaticCarcinoma

The expression of SPARC protein was examined in 25 primary pancreaticadenocarcinoma tissues by immunohistochemical labeling with ananti-SPARC monoclonal antibody. In 19 (76%) of 25 cases, moderate (++)to strong (+++) SPARC expression was found in the peritumoral stromalcells, presumably fibroblasts, and positive immunolabeling wasidentified as dark brown granules throughout the cytoplasm (FIG. 2). Inthese cases, the expression was most pronounced in the stromalfibroblasts immediately adjacent to the neoplastic epithelium, whereasthe staining was weak or absent in the stroma distant from theinfiltrating carcinoma. Immunolabeling of SPARC was also observed inneoplastic epithelium in 8 (32%) of 25 cases, but the labeling was weakand focal, with the exception of a single case in which 50% of theneoplastic cells strongly labeled. In the remaining 17 cases (68%),neoplastic cells did not label for SPARC throughout the tumor (FIG. 2).The immunoreactivity in normal ductal epithelium was variable amongcases; some normal ductal cells showed weak cytoplasmic staining butothers did not. These immunohistochemical findings suggest that theincreased SPARC tags detected in the SAGE libraries of the primarypancreatic cancer tissues originated primarily from stromal fibroblasts.

Example 4 Methylation Analysis of SPARC Gene in Pancreatic Cancer

We next analyzed the methylation status of the SPARC gene in a panel of17 pancreatic cancer cell lines. SPARC has a relatively CpG-richsequence spanning from exon 1 to intron 1 (GC content of 64%, ratio ofCpG to GpC of 0.6, and a length of 279 bp), which fulfills the criteriaof CpG island (FIG. 3A). Using MSP, we found that the SPARC CpG islandwas aberrantly methylated in 16 (94%) of the 17 pancreatic cancer celllines (FIG. 3B). The methylation status of SPARC correlated with itsexpression, and 15 (94%) of the 16 cell lines with aberrant methylationdemonstrated absent mRNA expression. By contrast, methylated alleleswere not identified in fibroblasts, in a non-neoplastic ductal cell line(HPDE), or in a pancreatic cancer cell line (PL9) with high mRNAexpression (P=0.004).

To confirm that DNA methylation is a mechanism for the silencing ofSPARC, we treated 8 pancreatic cancer cell lines harboring SPARCmethylation with the demethylating agent 5Aza-dC. The SPARC mRNAexpression was restored in 7 of the 8 cell lines after 5Aza-dC treatment(FIG. 3C). In one cell line (Hs766T); however, 5Aza-dC treatment did notrestore the SPARC expression. Furthermore, treatment of Hs766T with thehistone deacetylase inhibitor trichostatin A (TSA) or with a combinationof 5Aza-dC and TSA did not induce the SPARC expression (data not shown).These results suggest that other mechanisms besides DNA methylation andhistone deacetylation may be involved in the silencing of SPARC in thiscell line.

The methylation status of SPARC was also analyzed in a panel of 24xenograft tumors established from human primary pancreatic carcinomasand compared it to methylation patterns in 10 normal pancreatic ductalepithelia selectively microdissected by LCM. Aberrant methylation ofSPARC was detected in 21 (88%) of the 24 pancreatic xenografts (FIG.3D), whereas none of the 10 normal ductal epithelium samples displayedmethylated alleles (FIG. 3E). These results confirm the abnormalmethylation pattern of SPARC in primary pancreatic carcinomas as well asin pancreatic cancer cell lines.

Example 5 Effect of SPARC on Proliferation of Pancreatic Cancer Cells

Since SPARC is a secreted protein and has multiple biological functions,the altered patterns of SPARC expression in pancreatic cancer cells andstromal fibroblasts could affect tumor progression at the site oftumor-host interface. Based on the expression data, we hypothesized thatSPARC protein is secreted from stromal fibroblasts within invasivepancreatic carcinoma. To test this hypothesis, we measured the SPARCconcentration in conditioned media from three pancreatic cancer celllines (AsPC1, BxPC3, and Panc1) and fibroblasts derived from pancreaticcancer (panc-f5) by ELISA. The amount of SPARC secretion was negligible(0-30 ng/ml) in media from AsPC1 and BxPC3 with no detectable mRNAexpression, and a slightly higher secretion of SPARC protein (˜100ng/ml) was found in Panc1 with detectable mRNA expression. The highestSPARC secretion (˜1400 ng/ml) was identified in the fibroblast cultures.These results demonstrate a correlation between SPARC mRNA expressionand the amount of SPARC secretion in vitro.

The effect of exogenous SPARC protein on growth of pancreatic cancercells in vitro was also examined. We treated two pancreatic cancer celllines (AsPC1 and Panc1) with purified SPARC protein and counted thenumber of cells after 72 hours. Treatment with exogenous SPARC (10μg/ml) significantly suppressed the growth of AsPC1 cells by ˜27%(5.8±0.8 versus 4.2±0.3 (×10⁴ cells), P=0.001) (FIG. 4). Similarly,exposure of Panc1 cells to SPARC (10 μg/ml) resulted in growthinhibition by ˜30% (5.0±0.4 versus 3.5±0.4 (×10⁴ cells), P<0.0001) (FIG.4). Thus, these results suggest that exogenous SPARC protein hasgrowth-suppressive activity on pancreatic cancer cells.

Example 6 Serum SPARC Levels in Patients with Pancreatic Disease

The concentration of SPARC protein was measured in serum samples from 20patients with pancreatic adenocarcinoma, 20 patients with benignpancreatic disorders, and 20 healthy individuals by ELISA. There was nosignificant difference in the mean SPARC levels among these three groups(data not shown).

Example 7 Effects of Tumor-Stromal Interactions on SPARC Expression inFibroblasts

To elucidate the relationship between tumor-host interactions andtranscriptional regulation of SPARC in stromal fibroblasts, the SPARCmRNA expression was compared in three primary fibroblast culturesderived from different histological types of pancreatic tissues. Usingsemi-quantitative RT-PCR, we found that fibroblasts derived from chronicpancreatitis tissue (panc-f1) and those from non-cancerous pancreatictissue from a patient with pancreatic cancer (panc-f3) showed weakerexpression of SPARC mRNA compared to fibroblasts derived from pancreaticcancer tissue (panc-f5) (FIG. 5A). These results, together with theimmunohistochemical finding of SPARC expression localized to theperitumoral stroma, have led us to hypothesize that SPARC expression inthe stromal fibroblasts is modulated by interactions with tumor cells.To directly test this hypothesis, we utilized a co-culture system inwhich fibroblasts (panc-f3) and pancreatic cancer cells (CFPAC1) cancommunicate via soluble factors. SPARC mRNA expression in panc-f3 wasmarkedly (˜4,6-fold) augmented when these cells were co-cultured withpancreatic cancer cells (FIG. 5B). Thus, the SPARC transcription in thefibroblasts can be up-regulated in response to soluble mediatorssecreted by pancreatic cancer cells.

Because several growth factors such as TGF-β are known to induce theSPARC expression in fibroblasts (Wrana et al., 1991; Reed et al., 1994),and because TGF-β is one of the major secreted proteins highly expressedby pancreatic cancer cells (Friess et al., 1993), we examined the effectof TGF-β on SPARC expression in fibroblasts (panc-f3). When thefibroblasts were incubated with TGF-β (5 ng/ml) for 24 hours, the SPARCmRNA expression was increased by ˜3,3-fold (FIG. 5C), indicating thatTGF-β may be a candidate of tumor-derived factors that stimulate thetranscription of SPARC in stromal fibroblasts in a paracrine fashion. Wealso treated two pancreatic cancer cell lines with differing endogenousSPARC expression (AsPC1 with no mRNA expression and Panc1 withdetectable expression) with TGF-β (5 ng/ml). After treatment, a slightincrease (˜1,5-fold increase) in the SPARC mRNA expression was observedin Panc1, whereas the transcript remained undetectable in AsPC1 (datanot shown).

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1. A method for diagnosing cancer, comprising the detection of amethylated SPARC nucleic acid molecule or a variant thereof in a samplefrom a subject.
 2. The method of claim 1 wherein the presence of amethylated SPARC nucleic acid molecule is compared to a sample from asubject without cancer.
 3. The method of claim 1 wherein the sample isobtained from a mammal suspected of having a proliferative cell growthdisorder.
 4. The method of claim 1 wherein the sample is obtained from amammal suspected of having a pancreatic cancer.
 5. The method of claim1, wherein a methylated SPARC nucleic acid molecule comprises a sequencecorresponding to SEQ ID NO: 1 (FIG. 6).
 6. The method of claim 1,wherein a methylated SPARC nucleic acid molecule comprises a sequencehaving at least about 80% sequence identity to a molecule identified inSEQ ID NO: 1 (FIG. 6).
 7. The method of claim 1, wherein a methylatedSPARC nucleic acid molecule comprises a sequence having at least about90% sequence identity to a molecule identified in SEQ ID NO: 1 (FIG. 6).8. The method of claim 1, wherein a methylated SPARC nucleic acidmolecule comprises a sequence having at least about 95% sequenceidentity to a molecule identified in SEQ ID NO: 1 (FIG. 6).
 9. Themethod of claim 1, wherein the nucleic acid molecule is expressed atleast a lower level in a patient with cancer as compared to expressionlevels in a normal individual.
 10. The method of claim 1, wherein thenucleic acid molecule is expressed at least about 5 fold lower in apatient with cancer as compared to expression in a normal individual.11. The method of claim 1, wherein the nucleic acid molecule isexpressed at least about 10 fold lower in a patient with cancer ascompared to expression in a normal individual.
 12. The method of claim1, wherein the cancer is a pancreatic cancer.
 13. The method of claim 1,wherein the subject sample is obtained from a mammalian patient.
 14. Themethod of claim 1, wherein the subject sample is obtained from a humanpatient.
 15. A method of treating a patient with cancer wherein thecancer cells contain a methylated SPARC nucleic acid molecule comprisingthe administration to the patient a therapeutically effective amount ofdemethylating agent.
 16. A method of claim 15, wherein the demethylatingagent is 5-aza-cytidine.
 17. A method of claim 1, wherein the method ofdetecting a methylated SPARC nucleic acid comprising methylationspecific polymerase chain reaction (MSP).
 18. A method for detecting amethylated CpG-containing SPARC nucleic acid molecule comprising:contacting a nucleic acid-containing specimen with bisulfite to modifyunmethylated cytosine to uracil; contacting the SPARC nucleic acidmolecule with oligonucleotide primers that discriminate betweenmethylated and unmethylated CpGs; and detecting the methylated CpGs inthe nucleic acid.
 19. The method of claim 18, further comprisingamplifying the CpG-containing nucleic acid in the specimen by means ofthe oligonucleotide primers.
 20. The method of claim 19, wherein theamplifying step is the polymerase chain reaction (PCR).
 21. The methodof claim 18, wherein the CpG-containing nucleic acid is in a promoterregion.
 22. The method of claim 21, wherein the promoter is a tumorsuppressor gene promoter.
 23. The method of claim 18, wherein thespecimen is from a tissue selected from the group consisting ofpancreas, brain, colon, urogenital, lung, renal, hematopoietic, breast,thymus, testis, ovarian, and uterine.